A thermophilic bacterium, Bacillus sp. 4, newly isolated from Alangüllü thermal spring (Aydın, Turkey), showed a cell-associated esterase activity. Culture conditions in the growth and esterase production by the Bacillus sp. 4 were investigated using partially modified Thermus medium at different pHs (pH 5.00–9.00) and temperatures (50–70°C). The optimal growth and esterase production was obtained at pH 6.00 and 65°C. The maximal esterase production was obtained in the mid-stationary phase, and its activity was either intracellular or membrane associated. Optimum pH and temperature for esterase activity were 6.00 and 65°C, respectively. After 1 and 10h incubation at 65°C, the enzyme exhibited approximately 70 and 50% of its original activity, respectively. After 100h incubation at 40°C, the original activity of the enzyme was almost protected (83%). The esterase activities were about 99, 100, 100 and 81% of their original values after 1h incubation at pH 4.00, 6.00, 8.00 and 10.00, respectively. When the pNPB (C 4 ) was used as substrate, the Michaelis–Menten constant (K m ) and maximum velocity for the reaction (V max ) of esterase were 62.89μM and 833.33U/mg protein, respectively. Phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor, strongly inhibited the esterase activity, whereas β-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. The molecular mass (M r ) of the esterase was estimated to be 81.9kDa using SDS-PAGE. These results strongly suggest the presence of a single enzyme responsible for pNPB activity in the crude enzyme extract. Of all substrates (C 2 –C 16 ) tested, the highest activity was towards pNPB, whereas no activity was observed on pNPP (C 16 ).